Background
Sepsis is an overreaction of the host to severe infection, which mainly causes dysfunction of vital organs and is one of the common causes of death in hospitals, especially in the intensive care unit (ICU) [
1]. The lung is the first organ that responds to sepsis stimulation, and about 50% of acute lung injury (ALI) is related to sepsis [
2]. ALI is a pulmonary manifestation of systemic inflammation, which is mainly characterized by severe hypoxemia and immune cells infiltration in lung, which often result in death because of acute respiratory distress [
3]. The current treatment of ALI is based on antibiotics and supportive therapy, while the curative effect is very limited, and the mortality rate after treatment remains high. Therefore, research on effective biomarkers is essential for early diagnosis and timely treatment of ALI.
Long noncoding RNAs (lncRNAs) are endogenous RNAs with a length greater than 200 bp. Although lack of protein-coding ability, lncRNAs are of great significance for treating malignant tumors [
4]. In addition, multiple lncRNAs have shown excellent resistance to sepsis-induced organ damage. For example, Wang et al. showed that lncRNA NEAT1 eases the lipopolysaccharide (LPS)-induced myocardial damage in mice by inhibiting TLR2/NF-κB [
5]. Besides, Jiang et al. found that lncRNA HOTAIR alleviates ALI in septic rats by inhibiting the miR-34a/Bcl-2 axis [
6]. Small nucleolar RNA host gene 16 (SNHG16) is a type of lncRNA located on chromosome 17q25.1. Studies have shown that SNHG16 serves as an oncogene in non-small cell lung cancer (NSCLC) [
7]. A recent study indicated that SNHG16 promoted TLR4 expression by targeting miR-15a/16, thus repressing the anti-inflammatory effects mediated by miR-15a/16 [
8]. More importantly, knocking down SNHG16 inhibits the CCL5 expression by up-regulating miR-146a-5p, thereby mitigating LPS-induced lung cell damage, which is helpful for the diagnosis and treatment of acute pneumonia [
9]. Additionally, SNHG16 aggravates LPS-induced acute pneumonia in A549 cells through modulating miR-370-3p/IGF2 axis [
10]. Therefore, those studies suggest that SNHG16 makes a great contribution in LPS-mediated inflammation, especially in lung injury.
MicroRNAs (miRNAs) are small double-stranded RNAs of 19–25 nucleotides in length. They regulate post-transcriptional gene expression through translation inhibition or degradation enhancement of messenger RNAs (mRNAs), which affects biological processes such as cell proliferation, differentiation and apoptosis [
11]. MiRNAs are not only abnormally expressed in lung cancer, but also contribute to regulating other lung diseases. For instance, some scholars have found that miR-150 reduces LPS-induced ALI by targeting AKT3 or inhibiting the JNK/NF-κB pathway [
12]. What’s more, Ju et al. stated that miR-27a reduces inflammatory stimulation by inactivating TLR4/MyD88/NF-κB, thereby relieving LPS-induced ALI in mice [
13]. MiR-128-3p is a newly discovered miRNA in recent years. Studies by Pan et al. illustrated that miR-128-3p is knocked down in lung cancer tissues and cell lines, which is helpful for the diagnosis of early lung cancer [
14]. Moreover, miR-128-3p facilitates the protection of dexmedetomidine against ALI in sepsis mice by dampening MAPK14 [
15]. Hence, miR-128-3p has the potential effect in preventing lung injury.
High Mobility Group Box (HMGB), including HMGB1, HMGB2, HMGB3 and HMGB4, is an extracellular endogenous molecule released after tissue injury [
16]. HMGB3 is mainly distributed in the nucleus, cytoplasm and chromosomes, and has 85–89% homology with HMGB1 and HMGB2 [
17]. Among the four members of HMGB, HMGB1 is considered as a potential therapeutic target for sepsis, and glycyrrhizin protects rats from sepsis-induced damage by preventing the HMGB1-mediated inflammation [
18]. Ueno et al. found that HMGB2 was upregulated in LPS-treated mice, and it was also positively expressed in the epithelial lining fluid and serum of ARDS/ALI patients [
19]. HMGB4 is expressed by neuronal cells and affects the expression of genes involved in neural differentiation [
20]. As for HMGB3, it plays a carcinogenic role in glioblastoma [
21] and NSCLC [
22], while its effect on sepsis-related diseases is scarcely reported.
Overall, the present studies indicate that SNHG16 is involved in lung injury by targeting miRNAs and regulating downstream molecules, and miR-128-3p has potential effects in alleviating ALI in septic mice. Interestingly, our study found that SNHG16 and HMGB3 were up-regulated in sepsis model with ALI, while miR-128-3p was downregulated. Therefore, we speculate that there’s a regulatory axis of SNHG16/miR-128-3p/HMGB3 in sepsis-mediated ALI.
Methods
The rat model
Total Eighty male Sprague–Dawley rats (180–220 g, 6–8 weeks old) were purchased from the Animal Experiment Center of Huazhong University of Science and Technology (Wuhan, China). The rats were randomly divided into four groups (n = 20 in each group). The rats were maintained at 22–24 °C with 50–60% humidity and 12 h of light/dark cycle for one week, during which adequate food and water were given, and then cecal ligation and puncture (CLP) was performed to induce sepsis in rats. Preoperative fasting was applied to the rats. Afterward, pentobarbital sodium (50 mg/kg) was injected intraperitoneally to anesthetize the rats. After disinfection, the abdominal skin was incised for about 2 cm, and the cecum was gently pulled out with sterile forceps. The roots of the cecum were ligated with a 3–0 silk thread, and the head and tail of the cecum were perforated with a 20-G sterile needle. The two perforations were about 1 cm apart. Subsequently, a small amount of stool was gently squeezed out, and the cecum was repositioned in the abdominal cavity, and then the muscle, peritoneum and skin were sutured. Immediately after the operation, the rats were resuscitated by the subcutaneous injection of saline. E. coli counts in the bronchoalveolar lavage fluid (BALF) and peritoneal fluid were determined. The sham group did not undergo CLP, and the remaining steps were the same as the experiment group. In the CLP + sh-NC and CLP + sh-SNHG16 groups, rats were injected with 100 µl sh-NC or sh-SNHG16 (10 µM) (RiboBio Co., Ltd, Guangzhou, China) through the tail vein 48 h before CLP was performed. Finally, the rats were euthanized and the lung tissues were obtained and preserved in liquid nitrogen at − 196 °C for follow-up experiments. This experiment was carried out by observing the "Guidelines for the Care and Use of Laboratory Animals" (NIH Publication No. 85-23, 2011) published by the National Institutes of Health. All of the animal procedures had been approved by the medical ethics committee of Luoyang Central Hospital [approve number: SCXK(E)2016-0009] and also carried out in compliance with the ARRIVE guidelines (
http://www.nc3rs.org.uk/page.asp?id=1357).
Cell culture and transfection
Human normal lung epithelial cell line BEAS-2B was purchased from American Type Culture Collection (ATCC, Rockville, MD, USA) and cultured in the DMEM medium (Thermo Fisher HyClone, Utah, USA) containing 5% FBS (Thermo Fisher Scientific, MA, USA) at 37 °C with 5% CO2. Then, the cells were incubated with LPS (Sigma, Shanghai, China) at different doses (100 ng/mL) for 24 h.
The well-grown BEAS-2B cells were taken and inoculated into 6-well plates at 5 × 105/well. When the cells reach a fusion rate of 80–90%, they were transfected with 100 μL SNHG16 overexpression (SNHG16)/knockdown (sh-SNHG16) plasmids and miR-128-3p mimics (miR-128-3p) (300 nM) following the Lipofectamine 2000 reagent instructions (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA). The untreated cells served as control.
Quantitative reverse transcription PCR (qRT-PCR)
TRIzol (Invitrogen, Carlsbad, CA, USA) method was adopted to collect the total RNA from BEAS-2B cells and rat lung tissues, and the RNA was reversely transcribed into cDNA by referring to the instructions of the TOYOBO Reverse Transcription Kit (TOYOBO, Osaka, Japan). The reaction conditions for RT includes: total RNA (2 μg in DEPC H
2O (11 μL) and 14 μL oligo (DT) (10 μM) were mixed, 70 ℃ for 10 min, 1 ℃ for 1 min; then the first strand cDNA 2 μL, forward primer 2 μL, reverse primer 2 μL, dNTP (2 mm) 4 UL, 10 × PCR buffer 5 μL, Taq enzyme 1 μL were mixed, 42 ℃ for 50 min, 70 ℃ for 15 min to terminate the reaction, incubate at 37 ℃ for 20 min to degrade the residual RNA. qRT-PCR was implemented with the SYBR® Premix Ex Taq™ II Kit (Takara, Otsu, Japan), with GAPDH as the endogenous control for SNGG16 and HMGB3, while U6 was for the internal control of miR-128-3p. The fluorescent quantitative PCR system was 25 μL, containing 2 μL cDNA template, 250 nmol/L upstream and downstream primers and 12.5 μL 2 × SYBR® Premix. The reaction condition includes: 95 °C for 10 min, 40 cycles of 95 °C for 15 s and 60 °C for 1 min, 40 cycles. The primer sequences were shown in Table
1. The relative expression was calculated with the 2
−ΔΔCT method.
Table 1
Primer sequence of each molecule
SNHG16 | Forward: 5′-GTTCCTCTAAAAAAAAGCGCCATGCGTTCT-3′ |
Reverse: 5′-CATTTCAGTTTACAATCCTTGCAGTCCC-3′ |
miR-128-3p | Forward: 5′-GACTGCCGAGAGCGAGCG-3′ |
Reverse: 5′-GACGCCGAGGCACTCTCTCCT-3′ |
HMGB3 | Forward: 5′-ATTCGGAATTCCGTATCTGGCCTTTTGAC-3′ |
Reverse: 5′-CGGTTACTCGGCTTACGCTTGGACTG-3′ |
GAPDH | Forward: 5′-TGGTTGAGCACAGGGTACTT-3′ |
Reverse: 5′-CCAAGGAGTAAGACCCCTGG-3′ |
U6 | Forward: 5′-GTGCAGGGTCCGAGGT-3′ |
Reverse: 5′-CTCGCTTCGGCAGCACA-3′ |
Hematoxylin–eosin (HE) staining
The lung, liver, kidney and spleen tissues of the rat described in method 2.1 was fixed with 4% paraformaldehyde for 24 h, paraffin-embedded and sectioned (4 μm). Then, the sections were dehydrated with gradient alcohol, cleared with xylene, and mounted with resin. Then the sections were respectively stained with hematoxylin and eosin solution (Beyotime, Shanghai, China). Finally, the pathological changes of lung tissue were monitored with the microscope (Olympus BX 53 microscope, Tokyo, Japan).
Immunohistochemistry (IHC)
The lung tissues of sepsis rats were conventionally paraffin-embedded, sectioned (5 μm), dewaxed with xylene, hydrated with gradient alcohol and blocked with 3% H2O2 for 10 min to inactivate the endogenous peroxidase. After the sections were blocked with 5% bovine serum albumin for 20 min, they were incubated with the primary antibody Anti-Caspase-3 antibody (1:100, ab32351, Abcam, MA, USA) or anti-MPO antibody (1:100, ab208670, Abcam, MA, USA) at 4℃ overnight. After the sections were washed by PBS for 3 times (10 min each time), the secondary antibody Goat Anti-Rabbit IgG (1:500, ab150077, Abcam, USA) was added dropwise and incubated with the sections at room temperature (RT) for 20 min. Then, the sections were dehydrated, cleared, and fixed on a glass slide with neutral resin. At last, the sections were observed with a Nikon microscope (Nikon, Japan).
Wet-to-dry (W/D) method
The middle lobe of the right lung of experimental rats was taken, and the wet weight (W) was recorded after the surface water was sucked dry. Then the right middle lobe of the lung was heated in a constant temperature oven at 80 °C for 48 h to constant weight, weighed again and recorded the as the dry weight (D). The pulmonary edema index = W/D.
Arterial blood gas analysis
0.5 mL arterial blood of the rats was drawn from the abdominal aorta of a sepsis rat with a syringe supplemented with heparin and immediately injected into a blood gas analyzer (Beckman Coulter, Inc., USA) immediately to determine the arterial partial pressure of oxygen (PaO2), carbon dioxide (PaCO2) and pH value.
Enzyme-linked immunosorbent assay (ELISA)
The rat lung tissues were collected, then 200 μL PBS was used for tissue homogenate (per 100 mg tissue). The culture medium of BEAS-2B cells after treatments was also collected by centrifugation for removing cells or debris. The levels of IL-1β (RLB00 or DLB50), IL-6 (R6000B or HS600C), and TNF-α (RTA00 or HSTA00E) in CLP rat lung homogenate and culture medium were determined with ELISA kits following the supplier's guidelines (R&D Systems, Minneapolis, MN, USA). Then, the OD was observed with a Power Wave microplate reader (Bio-TEK, USA) at 450 nm.
Immunofluorescence
BEAS-2B cells were transfected with SNHG16 and/or miR-128-3p mimics. The stably-transfected cells were seeded in 24-well plates (1 × 105 cells per well). 24 h later, the cells were fixed with 4% paraformaldehyde at 25 °C for 30 min, and then permeabilized with 0.3% Triton X-100 for 20 min. Followed by that, the cells were blocked with 5% BSA (at room temperature for 1 h), and incubated with primary anti-HMGB3 antibody (ab75782, Abcam, USA) at 4 °C for 12 h. The next day, the cell were washed by PBS and incubated with Goat polyclonal Secondary Antibody to Rabbit IgG—H&L (Alexa Fluor® 488) at 4 °C for 1 h. After being washed with PBS, DAPI solution (10 μg/mL) was used for staining the nuclear for 10 min in the dark. Finally, a confocal laser scanning microscope (BIO-RAD, Hemel Hempstead, UK) was used for observing the cell images.
Western Blot (WB)
The total protein of lung tissues and BEAS-2B cells were extracted and then quantified with bicinchoninic acid assay (Pierce, Rockford, USA). Then, it was resolved on 10% polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride (PVDF) membranes. Afterward, the PVDF membranes were blocked with 5% skim milk at RT for 1 h and incubated with the primary Anti-NF-κB p65 (phospho S276) antibody (1:1000, ab222494, Abcam, USA), Anti-NF-κB p65 antibody (1:1000, ab16502, Abcam, USA), Anti-MAPK p38 (phospho T180) antibody(1:1000, ab178867, Abcam, USA), Anti-MAPK p38 antibody(1:1000, ab31828, Abcam, USA), Anti-iNOS antibody(1:1000, ab178945, Abcam, USA), Anti-COX2 antibody (1:1000, ab179800, Abcam, USA), Anti-HMG4 antibody (1:1000, ab75782, Abcam, USA), Anti-β-actin antibody (1:1000, ab8224, Abcam, USA) and Anti-GAPDH antibody (1:1000, ab181602, Abcam, USA) overnight at 4 °C. Subsequently, the membranes were washed twice with TBST, and then incubated with the fluorescein-labeled secondary Goat Anti-Rabbit IgG (1:3000, ab150077, Abcam, USA) at RT for 1 h. After that, the membranes were rinsed 3 times. The protein bands were examined by the ECL WB kit (Amersham Biosciences, UK). The primary images of the blots were shown in the Additional file
1: Fig. S1, Additional file
2: Fig. S2 and Additional file
3: Fig. S3.
Dual-luciferase reporter assay
The BEAS-2B cells were inoculated in 48-well plates at 5 × 105/well and cultured to 70% confluence. Then, the cells were transfected with SNHG16-WT, SNHG16-MUT, HMGB3-WT, HMGB3-MUT (0.5 μg) and miR-128-3p mimics and their negative controls (300 pmol) with the liposome 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA). After 48 h, the luciferase activity was monitored as per the supplier's instructions (Promega, Madison, WI, USA).
RNA immunoprecipitation (RIP) assay
RIP assay was carried out with the Magna RipRNA binding protein IP kit (Millipore, Bedford, MA, USA) following the kit’s protocol. The complete RIP lysis buffer was employed to lyse BEAS-2B cells, and then 100 μL of whole-cell extract was incubated with the RIPA buffer containing magnetic beads coupled with the human anti-Argonaute2 (Ago2) antibody (Abcam, Shanghai, China) at 4 °C for 6 to 8 h. Immunoglobulin G (IgG) antibody served as a negative control. Finally, RNA was extracted with the TRIzol reagent (Invitrogen, Carlsbad, CA, USA), reversely transcribed into cDNA, and analyzed by qRT-PCR.
3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay
Stably transfected BEAS-2B cells were inoculated in 96-well plates at 2 × 104/well and incubated at 37 °C with 5% CO2 for 24 h. Different factors were administered to treat the cells. The blank control group was supplemented with an equal volume of PBS, and 5 repetitive wells were set in each group. After culturing for 24 h, the cells were incubated with 20 μL MTT solution (5 mg/mL, Beyotime, Shanghai, China) at 37℃ for 4 h. After that, the supernatant was aspirated and DMSO was added to lyse the cells. After the crystals were dissolved, the OD value of each well was observed with a microplate reader (Bio-Rad, Hercules, CA, USA) at 570 nm.
Flow cytometry
AnnexinV/7-AADA apoptosis Detection Kit (Southern Biotechnology, Birmingham, Al, USA) was adopted to determine cell apoptosis following the kit instructions. The treated BEAS-2B cells were trypsinized and centrifuged, and then washed twice with cold PBS. Afterward, the cells were double-stained with AnnexinV and propidium iodide (PI) and incubated in the dark at RT for 15 min. Finally, the FACScan flow cytometer (FCM; Bechman Coulter, CA) was used to analyze apoptosis.
Statistical analysis
GraphPad Prism 8 (GraphPad Software, Inc., city, state) was employed to analyze statistical differences. The measurement data were presented as mean ± standard deviation (\({\overline{\text{x}}} \pm {\text{s}}\)). t test was used for pairwise comparison, and the comparison of multiple samples was made by one-way ANOVA. P < 0.05 indicated statistical significance.
Conclusions
Sepsis can cause life-threatening multiple organ dysfunction, of which ALI and acute respiratory distress are the earliest and most common symptoms. The main pathological manifestations of ALI are inflammatory cell infiltration, lung epithelial cell damage, endothelial cell destruction, pulmonary interstitial congestion and edema [
23]. Due to the complex pathological mechanism and the rapid progress of ALI, there is still a lack of effective clinical countermeasures. Our study proved that SNHG16 is up-regulated in septic ALI rats, and down-regulating SNHG16 eases lung injury by modulating the miR-128-3p/HMGB3 axis.
LncRNAs are abundant in mammals. They modulate gene expression at the transcriptional and post-transcriptional levels and contribute to diversified biological processes such as chromatin remodeling, histone modification, and cell cycle regulation [
24]. Over the years, several studies have shown that lncRNAs intervene inflammation by modulating inflammatory mediators. Some scholars discovered that MALAT1 is up-regulated in rat tissues with septic lung injury, and knocking down MALAT1 ameliorates the lung injury in rats by attenuating the MAPK p38/NF-κB p65 signaling pathway [
25]. SNHG16 is a novel lncRNA, which was first studied by Christensen et al. Their findings showed that SNHG16 is up-regulated in colorectal cancer cell lines, and knocking down SNHG16 inhibits cell proliferation and migration, and induces apoptosis [
26]. Also, the carcinogenic effect of SNHG16 is proved in NSCLC [
22]. Besides, SNHG16 is abnormally expressed in other lung diseases, including ALI. For instance, Zhang et al. hold that SNHG16 up-regulates IGF2 by targeting miR-370-3p, thereby preventing LPS from inhibiting lung cell viability and promoting cell apoptosis and inflammation [
10]. Moreover, Zhou et al. confirmed that SNHG16 is up-regulated in the serum of ALI patients, and knocking down SNHG16 in the LPS-treated lung cell injury model inhibits the expression of CC motif chemokine ligand 5 (CCL5) by sponging miR-146a-5p, thereby reducing the inflammatory injury of lung cells [
9]. Thus, knocking down SNHG16 may be a new treatment for ALI. Fortunately, our research results suggested that knocking down SNHG16 abates sepsis-mediated lung injury and dysfunction, reduces pulmonary edema and inflammation, improves lung cell viability, and prevents cell apoptosis.
Accumulating researches have proved that miRNAs contribute to regulating sepsis-induced ALI. For example, Cao et al. reported that miR-145 targets TGFBR2 and alleviates sepsis-induced ALI [
27]. In addition, Pan et al. also showed that miR-124 reduces the ALI symptoms in septic mice by down-regulating MAPK14 and inactivating MAPK signaling pathway [
28]. The literature also revealed that miR-98 negatively regulates HMGA2 by inhibiting the NF-κB signaling pathway to protect mice from sepsis-mediated lung damage [
29]. Still, we are very curious whether miR-128-3p can treat sepsis-induced ALI by regulating the downstream genes. Ding et al. took the lead in research and proved that miR-128-3p promotes the SOD expression while down-regulates MPO and MDA in septic mouse tissues. Meanwhile, miR-128-3p enhances the protection of dexmedetomidine against ALI in sepsis mice by targeting and dampening MAPK14 [
15]. Above all, some studies have found that down-regulated SNHG16 inhibits HOXA7 by sponging miR-128-3p to prevent the development of glioblastoma [
30]. Here, we discovered that miR-128-3p is a vital downstream target of SNHG16 through bioinformatics, and we confirmed that miR-128-3p is negatively regulated by SNHG16. Meanwhile, the miR-128-3p mimic transfection represses the LPS-induced apoptosis and enhancement of inflammation and reduces lung injury.
Studies have shown that miRNAs bind to the 3′-UTR of their target mRNAs and reduce their expression and contribute to gene regulation. HMGB3, a multifunctional protein and a crucial downstream target of various miRNAs, is involved in the occurrence and progression of diversified malignancies. Yamada et al. found that the targeted inhibition of HMGB3 by miR-205-5p reduces the aggressiveness of prostate cancer [
31]. Some scholars have found that overexpressing miR-758 targeting HMGB3 inhibits the malignant biological behaviors and strengthens NSCLC cell apoptosis [
32]. However, whether HMGB3 plays a role in sepsis-induced organ inflammation remains elusive. Biological information analysis demonstrated that miR-128-3p targets and inhibits HMGB3, and overexpressing HMGB3 counteracts miR-128-3p. Namely, overexpressing HMGB3 partially eliminates the protective effect of miR-128-3p on LPS-damaged lung cells, promotes cell apoptosis, inhibits cell viability, and activates inflammation.
Overall, SNHG16 is up-regulated in sepsis-mediated ALI. In addition, inhibiting SNHG16 targets miR-128-3p and up-regulates HMGB3, thereby reducing lung injury, pulmonary edema and inflammation, and increasing lung cell viability and preventing cell apoptosis. Nevertheless, we only conducted male animal experiments in this study. In the future, more researches need to be conducted: (1) To confirm the regulatory axis of SNHG16/miR-128-3p/HMGB3 in larger number of female and male rats; (2) To evaluated the expressions of SNHG16/miR-128-3p/HMGB3 in ALI patients with sepsis.
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